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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Identification of Features Regulating OST1 Kinase Activity and OST1 Function in Guard Cells^1,[W]

Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette cedex, France (C. Belin, P.-O.F., C. Bourbousse, J.G., H.B.-B., S.T.); Institut Européen de Chimie et Biologie, 33607 Pessac, France (S.C., J.-M.S.); and Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Laboratoire des Echanges Membranaires et Signalisation, 13108 St. Paul lez Durance cedex, France (A.V.)

The phytohormone abscisic acid (ABA) mediates drought responses in plants and, in particular, triggers stomatal closure. Snf1-related kinase 2 (SnRK2) proteins from several plant species have been implicated in ABA-signaling pathways. In Arabidopsis (Arabidopsis thaliana) guard cells, OPEN STOMATA 1 (OST1)/SRK2E/SnRK2-6 is a critical positive regulator of ABA signal transduction. A better understanding of the mechanisms responsible for SnRK2 protein kinase activation is thus a major goal toward understanding ABA signal transduction. Here, we report successful purification of OST1 produced in Escherichia coli: The protein is active and autophosphorylates. Using mass spectrometry, we identified five target residues of autophosphorylation in recombinant OST1. Sequence analysis delineates two conserved boxes located in the carboxy-terminal moiety of OST1 after the catalytic domain: the SnRK2-specific box (glutamine-303 to proline-318) and the ABA-specific box (leucine-333 to methionine-362). Site-directed mutagenesis and serial deletions reveal that serine (Ser)-175 in the activation loop and the SnRK2-specific box are critical for the activity of recombinant OST1 kinase. Targeted expression of variants of OST1 kinase in guard cells uncovered additional features that are critical for OST1 function in ABA signaling, although not required for OST1 kinase activity: Ser-7, Ser-18, and Ser-29 and the ABA-specific box. Ser-7, Ser-18, Ser-29, and Ser-43 represent putative targets for regulatory phosphorylation and the ABA-specific box may be a target for the binding of signaling partners in guard cells.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christophe Belin (belin{at}isv.cnrs-gif.fr).

^[W] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.079327.

^* Corresponding author; e-mail belin{at}isv.cnrs-gif.fr; fax 33–1–69823768.

Received February 24, 2006; returned for revision May 31, 2006; accepted June 1, 2006.

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